Biotin intermediate



Patented Oct. 16, 1951 BIOTIN INTERMEDIATE Stanton A. Harris, Westfield, Ralph Mozingo, Elizabeth, Glen E. Arth, Rahway, and Karl Folkers, Plainfield, N. J assignors to Merck & 00., Inc., Rahway, N; J., acorporation of New Jersey No Drawing. Application December 15, 1945, Serial No. 635,424

12 Claims. (Cl. 260332.2)

This invention is concerned generally with It is now found that this compound can be synthesized by reactions indicated as follows:

The reactions indicated above are conducted as follows: 2- (4-carboxy-butylidene) -3-keto-4- acylamido tetrahydrothiophene ester (1) is reacted with one molecular equivalent of hydrogen in the presence of a hydrogenation catalyst to produce the corresponding ester of 2-(4-car-' boxy-butyl) -3-keto 4 acylamido tetrahydrothiophene (2). This compound is then reacted with av member of the group which consists of hydrbxylamine, arylhydrazines and semicarbazide to produce the corresponding 3-keto substitution product (3); in the case of hydroxylamine, this product is an ester of 2-(4-carboxy-butyl) 3-oximino-4 acylamido tetrahydrothiophene. This product is reacted in the presence of a. hydrogenation catalyst and preferably in the pres- I H d NH y rogen cn-s-c'iwnmcoon In the above formulae, R represents an alkyl, aryl or aralkyl group, X a halogen, and YNHz a compound selected from the group consisting of hydroxylamine, arylhydrazines and semicarba- Zide.

H-S-PJ (CHzhC O OH ence of hydrogen whereupon a simultaneous hydrogenation and dehydrogenation occurs forming the 2-(4'-carboxy-butyl)-3-amino-4-acylamido-thiophene ester (4); alternatively the 3-keto substitution product can be treated with zinc in the presence of a mixture of an aliphatic organic acid and its anhydride, whereby the 2- (4' -carb oxy-butyl) -3 :4-diacylamido-thiophene ester (5) results; when these acylamido derivatives are treated with an aqueous alkaline solution, hydrolysis of the acyl groups occurs, yielding 2- (4'-carboxy-butyl) -3 4-diamino-thiophene (6) which, when reacted with a carbonyl halide produces the compound 2-(4'-carboxy-butyl) 3:4-ureido-thiophene (7).

The starting material, the 2 -(4-carbox y-bu tylidene) 3 keto-4-acylamido-tetrahydrothiophene ester can be obtained as indicated in general terms in J. A. C. S., 66, 1756-7 (1944) and as described in detail in co-pending applications, Serial Nos. 554,458; 554,449; 554,450; 554,451 and 554,452, all filed September 16, 1944, now Patents 2,466,232; 2,537,892; 2,537,893; 2,508,457, and 2,452,653, respectively. Inasmuch as the process according to this invention is applicable to commun s o e e e al c ass r es ed by Formula 1 above, it will be apparent that equivalent compounds within this general class can be substituted for the specific compounds mentioned in the following examples. Among these equivalent starting materials are: 2-(4'-carbomethoxy-butylidene) 3 keto-4-benzamldo-tetrahydrothiophene; 2-(4f-carboethoxy-butylidene) -3-keto-4- benzamido tetrahydrothiophene; 2 (4-carbobenzoxy butylidene) -3-keto-4-benzamido-tetrahydrothiophene; 2 (4 carbopropoxy butylidene) 3-keto-4-acetamido-tetrahydrothiophene; 2- (4' -carbophenoxy-butylidene) -3-keto-4-propamido-tetrahydrothiophene.

When these compounds are reacted with 1 molecular equivalentof hydrogen in the presence of a hydrogenation catalyst, the product obtained is the corresponding ester of 2-(4-carboxybutyl) 3 keto-4-acylamido-tetrahydrothiophene. This product can be reacted with hydroxylamine or a salt thereof; an arylhydrazine such as p-nitrophenyl hydrazine, 2,4-dinitrophenyl-hydrazine and the like; or semicarbazide to produce a compound of the formula:

wherein R represents an alkyl, aryl or aralkyl group and YN= is a radical of the class which consists of oximino, arylhydrazino and semicarbazido. This reaction can be carried out in aqueous solution, or if desired, in an aqueous or non-aqueous organic solvent, as for example, a lower aliphatic alcohol.

When this 3-ketosubstitution product is allowed to react in the presence of hydrogen and a hydrogenation catalyst, a simultaneous hydrogenation and dehydrogenation takes place, whereby the 3-substituent is reduced to an amino group and at the same time the tetrahydrothiophene ring is dehydrogenated to a thiophene ring, to produce the 2-(4-carboxy-butyl) -3-amino-4-acylamido-thiophene ester. It is presently preferred, where practicing the process according to this invention, to use a noble metal hydrogenation catalyst, particularly platinum or palladium. It is preferred that the catalyst be distributed upon a suitable carrier such-as charcoal, diatomaceous silica, barium sulfate and the like, suspe lded in;

hr la e' Sol nt a forv example). at r 4 or an organic solvent such as methanol, ethonal and the like.

The hydrogenation-dehydrogenation operation can also be conducted by allowing the 3-keto substitution product of the 2-( 4-carboxy-butyl) -3- keto-4-acylamido-tetrahydrothiophene ester to react with zinc in the presence of a mixture of an organic acid and its anhydride, whereby the group in the 3-position is simultaneously reduced and acylated to produce a 3-acylamido substitu ent and the tetrahydrothiophene ring is dehydrogenated to thiophene. Acids and anhydrides which can be employed in this reaction include acetic acid and its anhydride, propanoic acid and its anhydride and the like; it is presently preferred to employ the metal in the form of a finely divided powder.

When compounds of the formula:

RCO

111E I|\|IY' tH O (CH2).4 C 0 0 R. (9) wherein R represents .an alkyl, arylor aralkyl groupv and Y-=N= is a. radical of the class which consists of .ox-imino, arylhydrazino and semi-. carbazido,.are reactedwith a reducing agentaoe cording to the processes described in co-..pending applications Serial No. 635,426, filed December :15, 1945, and Serial No. 635,427, filed December 15, 1945, now Patents 2,487,051 and 23,60,225, respectively, the chief products obtained may be represented by the general formulae:

wherein has the, significance above-defined. In addition to these compounds, however, byproducts are obtained having the formula:

RCO R l NE NH r ln-fswnplcoon wherein R is an ,acyl group or hydrogen and R has the significance above-defined. These byproducts are produced by dehydrogenation of the tetrahydrothiophene ring which occurs simultaneously with the, hydrogenation reaction. Under certain conditions, the dehydrogenation reaction described above predominates, as for example: when 2- (4-carbomethoxy-butylidene) 3-(p-nitrophenyl-hydrazino) 4 -benzamido-tetrahydrothiophene is heated with zinc and;a mixture comprising acetic acid and acetic anhydride, or when said; compound is reacted with hydrogen at room temperature in the presence of acetic anhydride and Willstatter palladium, the main r duct s, 4' cerbqme hoxywhutyn.-a-acetami- -4-benzam do thion e ehe ac la do empounds. obtained by these. dehydrogenation reactions, namely the esters; of.

2- (4-carboxy-butyl) -3-amino-4-acylamid0-thio-l phene and 2- (4-carboxy-butyll -'=i4 diacylam1- do-thiophene can be hydrolyzed by heating with an aqueous solution of an alkali metal or an alkaline. earth metal hydroxide; it is presently pr b ferred to use aqueous barium hydroxide solution, to produce 2-(4-carboxy-butyl) -3:4-diaminothiophene. This compound can then be reacted with a carbonyl halide such as phosgene prefer ably at a temperature of the order of 0 C., and in an aqueous alkaline solution, containingan alkali metal hydroxide or an alkali metal salt of a weak acid such as sodium carbonate or the like to produce the desired 2-(4'-carboxy-butyl) -3:4

ureido-thiophene. I I

The following examples illustrate methods of carrying out the present invention but it is to be understood that these examples are given by way of illustration and not of limitation.

About 1.7 gms. of 2-(4-carbomethoxy-butylidene) -3-keto-4- benzamido tetrahydrothiophene which can be prepared as described in co-pending application, Ser. No. 554,452, filed September 16, 1944, is dissolved in about 200 cc. of methanol in which is suspended the palladium-charcoal catalyst obtained by previously reducing about gms. of charcoal-palladium-chloride mixture containing 5% by weight of palladium-chloride. This mixture is then shaken with hydrogen at-40 pounds per square inch pressure until one mole equivalent has been absorbed. This takes approximately 4 hours. The slurry is then filtered and the catalyst washed thoroughlywith methanol; the methanol solution is evaporated and the concentrated solution cooled and the crystals which form are filtered and dried to producev sub. stantially pure 2 (4' 7-. carbomethoxy-butyl)-. 3+ keto-4-benzamido-tetrahydrothiophene; M.

- Example 2 CaHrCO 11TH 0 tsflt we Hr-S-(JH(CH2)4COOCH3 cameo 1 m NOH tH t butyl) 3-oximino-4-benzamido tetrahydrothio-' phene separates and is recovered by filtrationand dried; M. P. -161" C. This oximiiio ester can be converted to the free acid by boiling with an aqueous methanol solution of sodium methoxide, which, upon acidification, yields a substantially pure formof the corresponding stereoisomer of 2-(4'-carboxy-butyl) -3-oximino 4 benzamido tetrahydrothiophene; M. P. 201 C.

A second stereoisomeric oxime is prepared as follows: the methanol solution obtained in Example 1 after filtration of the stereoisomeric racemate of 2-(4-carbomethoxy-butyl) -8-keto- 4-benzamido-tetrahydrothiophene (M. P. 114- 116 C.) is treated with an excess of hydroxylamine hydrochloride and sodium acetate and the mixture heated on a steam bath for about hour. The reaction mixture is diluted with water and the crude product which separates is fractionally crystallized from ethyl acetate toproduce a second stereoisomeric racemate of the oxime prepared above, which has a difierent crystal structure. This material is purified by recrystallization from methanol-water solutionlto produce a second stereoisomeric racemate of- 2-(4'-carbomethoxy-butyl) 3 -oximino- 4 -benzamido-tetrahydrothiophene; M. P. 152-l54 C.

Example 3 C|H|C O NH O JJIL- l Hz-S-CIKCHzhC O O CH:

CuHaCO NH NNH CcH4NOz tH t About 1.4 gms. of 2-(4'-carbomethoxy-butyl)- 3-keto-4-benzamido-tetrahydrothiophene is dissolved in about 50 cc. of methanol containing one drop of concentrated hydrochloric acid and the resulting solution is heated under reflux with one molecular equivalent of p-nitrophenyl-hydrazine for approximately /g hour. The reaction mixture is cooled and allowed to stand for approximately two days, whereupon the p-nitrophenol-hydrazone of the starting material crys; tallizes and is recovered by filtration and dried; M. P. 143-145 C.

Example 4 C035 0 NH NOH Zinc t v t H-- Acetic acid Acetic anhy. Hr-S H(CH2)4COOCH$ CcHs CO C O CH;

17TH NH F u H-S-C (CH2)4COO CH;

About 1.8 grams of 2- (4-carbomethoxy-butyl) 3 oximino 4-benzamido-tetraphydrothiophene' Example 5 CAEG Zinc NH N NH CtHiN O: Acetic Acid ll Acetic Anhy.

H-C 1 Heat CHr-S- CH(CH2)C 0 0 0H:

Cal-LC 0 C 0 CH4 rim NH o H benzamido tetrahydrothiophene (prepared as de scribed in co-pending application Serial No. No. 635,427, filed December 15, 1945, now Patent No. 2,460,225) is dissolved in about 125 cc.'o acetic anhydride containing 2 cc. concentrated. hydrochloric acid. 0.5 gm. of Willstatter palladium (prepared as described in Ber. 54, .123 (1921)) is added and the mixture is shaken for 4 days. The acid is neutralized with sodium acetate, and solvent is distilled off under a reduced pressure and at low temperature. The residual product is suspended in water and the organic material extracted therefrom by means of chloroform. The chloroform layer is then evaporated to produce a dark-brown viscous oil which is extracted with ether and the ether extract evaporated to produce a bright-yellow oil which partially crystallizes upon standing. The crystalline material is purified by recrystallization from methanol to produce nearly white crystals of 2 (4' carbomethoxy butyl) 3- acetamido-4 benzamido-thiophene; M. P. 159- 160 C.

Example 7 CaHsCO NH NOH (2) Acetic Anhydnda 0.11.00 coorn. cameo oocm About 1.2 gms. of 2-(4'-carbomethoxy-butylidene) 3- (p nitrophenyl hydrazino) 4- benzamido-tetrahydrothiophene is dissolved in a mixture of about 25 cc. of acetic acidand 25 cc. acetic anhydride. This solution is then heated on a steam bath with an excess of zinc dust for approximately 2 hours, at which time the yellow color of the solution disappears. The solution isthen filtered to remove unreacted zinc dust and evaporated to dryness under reduced pressure, and the residue dissolved in a mixture of chloroform and water; The chloroform-water solutions are filtered from a White insoluble product (which is the diacetate of phenylene diamine), the chloroform layer is evaporated to dryness and the; residue extracted with ether. Upon evaporation of the ether extract, 0.7 gm. of crystalline material is obtained which is purifled by recrystallization from methanol to produce pure 2 (4' carboxy butyl) 3 acetamido-4-benzamido thiophene; M. P. 159-160 C.

About. 0.9 gm. of 2-(4'-carbomethoxy-butylidenei 3 (p nitrophenylhydrazinoi 4- dm-s-i imonoro o 0 on.

tin-sixomncooom About one gram of the mixed oximes of 244- carbomethoiwbutylidene) 3-keto4-benzamido tetrahydrothiophene i dissolved in about 189 mL of methanol containing about 11 ml. of 3.3% hydrochloric acid. About 15 g. of 5% palladium on barium sulfate catalyst is added to this solution and the mixture shaken under three atmospheres of hydrogen for approximately 40 minutes. at. which time about 12 gms. additional catalyst is added. The mixture is shaken under hydrogen pressure for approximately 18 hours additional time and the catalyst is removed by centrifuga-e tion. Water and enough sodium acetate is added to neutralize the acid, and the solution is extracted with chloroform. The chloroform extract is evaporated to dryness and the residue is acetylated by heating with acetic anhydride on the steam bath. After evaporation of the excess acetic anhydride, the residue is dissolved in chloroform and washed with aqueous sodium bicarbonate. The chloroform extract is evaporated to dryness to produce approximately 0.8 m. crude material.

A sample of this crude material is hydrolyzed by heating with aqueous barium hydroxide solution and then reacted with phosgene. Microbiologi'cal assayof' this. product shows biotin corresponding to approximately 25% of 2'-(4'- Qarboxy-butyll 3 acetamido 4 benzamidotetrahydrothiophene originally present.

..The remainder of the residue crystallizes on standing, This. separated chromatographically 20. g.- of activated charcoal from acetone solution and the adsorbed material eluted with. chloroform. The chloroform extract is evaporatedtofdryness and the. residue is recrystallized.

"from a methanol-ether solution to yield about 0.17 g. of 2-(4'-carbomethoXy-butyl)-3-acetamidol-benzamido-thiophene; M. P. 156-160 C.

This case is a continuation-in-part of application Serial No. 554,455, filed September 16, 1944, now Patent No. 2,487,050, by the same inventors, entitled Chemical Compounds and Processes of Preparing the Same.

Modifications may be made in carrying out the present invention without departing from the spirit and scope thereof and the invention is to be limited only by the appended claims.

We claim:

1. The process which comprises reacting 2-(4- carbomethoxy butyl)-3-oximino-4-benzamidotetrahydrothiophene with hydrogen in the presence of a noble metal hydrogenation catalyst to produce 2-(4'-carbomethoxy-butyl)-3-amino-4 benzamido-thiophene.

2. The process which comprises reacting 2-(4- carbomethoxy butyl)-3-oximino-4-benzamidotetrahydrothiophene with hydrogen in the presence of palladium to produce 2-(4carbomethxy-butyl) -3-amino-4-benzamido-thiophene.

'3. The process which comprises reacting 2(4- 'carbomethoxy butyl)-3-(p-nitrophenylhydraz- 'ino) -4-benzamido-tetrahydrothiophene with hydrogen in the presence of palladium to produce 2 (4' carbomethoxy butyl) 3 amino- 4 ibenzamido-thiophene.

4. The process which comprises reacting 2-(4'- carbomethoxy butyl)3-oximino-4-benzamidotetrahydrothiophene with zinc in the presence of a mixture which comprises acetic acid and its anhydride to produce 2 (4' carbomethoxybutyl) -3-acetamido-4-benzamido-thiophene;

5. 2-(4'-carbomethoxy butyl)3acetamido- '4-benzamido-thiophene.

6. 2-(4-carbomethoxy-buty1) 3 amino-4- Ibenzamido-thiophene. I

7. The process of preparing 3-amino-4-acylamido-2-(4'-carboxy butyl) thiophene esters which comprises reacting 2-substituted tetrahydrothiophenes having the formula:

wherein R is a radical selected from the group which consists of alkyl, aryl and aralkyl radicals, YN= is a radical selected from the group which consists of oximino, arylhydrazino and semicarbazido radicals, and having a 2-position substituent selected from the class which consists of 4' carboalkoxy butyl, 4' carboaroxybutyl, 4-carboaralkoxy-butyl, 4-carboalkoxybutylidene, 4-carboaroxy-butylidene and 4- carboaralkoxy-butylidene radicals, with hydrogen in the presence of a noble metal hydrogenation catalyst.

8. The process of preparing 3-amino-4-acylamino 2-(4carboxy butyl) thiophene esters which comprises reacting z-substituted tetrahy drothiophenes having the formula:

RCO

NH l| |1'Y (la-O Hz-S-JIHz wherein R is a radical selected from the group which consists of alkyl, aryl and aralkyl radicals, YN= is a radical selected from the group which consists of oximino, arylhydrazino and semicarbazido radicals, and having a 2-position substituent selected from the class which consists of 4' carboalkoxy butyl, 4' carboaroxybutyl, 4-carboaralkoxy-butyl, 4-carboalkoxybutylidene, 4-carboaroxy-butylidene and 4 carboaralkoxy-butylidene radicals, with hydrogen in the presence of a palladium catalyst.

9. The process of preparing 3:4-diacylamido- 2 (4'carboxy butyl) thiophene esters which comprises reacting 2-substituted tetrahydrothiophenes having the formula:

wherein R is a radical selected from the group which consists of alkyl, aryl and aralkyl radicals, YN= is a radical selected from the group which consists of oximino, arylhydrazino and semicarbazido radicals, and having a 2-position substituent selected from the class which consists of 4' carboalkoxy butyl, 4' carboaroxybutyl, 4-carboaralkoxy-butyl, 4-carboalkoxybutylidene, 4'-carboaroxy-butylidene and 4- carboaralkoxy-butylidene radicals, with zinc, an aliphatic acid and an aliphatic acid anhydride.

10. 2 (4' carboalkoxy butyl) 3:4-diacylamide-thiophene.

11. 2-(4'-carboalkoxy-butyl) 3 amino-4- acylamido-thiophene.

12. N acylated 2 (4-carboalkoxy-butyl) 3 :4-diamino-thiophene.

STANTON A. HARRIS. RALPH MOZINGO. GLEN E. ARTH. KARL FOLKERS.

REFERENCES CITED The following references are of record in the file of this patent:

UNITED STATES PATENTS Number Name Date 2,373,136 Hoover Apr. 10, 1945 2,399,974 Avison May 7, 1946 OTHER REFERENCES Karrer, Organic Chemistry, p. 428, Nordeman Publ., N. Y., 1938.

Karrer, Helv. Chim. Acta., 27, 143 (1944). 

12. N - ACYLATE -2 - (4'' -CARBOALKOXY-BUTYL)3:4-DIAMINO-THIOPHENE. 